Wei Pei, Xiao Yongqiang, Xu Zhaorong, Chen Xiaodong, Jiang Qiong, Fu Yu, Yan Jianji, Chen Zhaohong, Luo Pengfei, Liu Huazhen
Burn & Wound Repair Department, Fujian Medical University Union Hospital, Fuzhou, Fujian, China.
Fujian Burn Institute, Fujian Medical University Union Hospital, Fuzhou, Fujian, China.
J Cell Mol Med. 2025 Jun;29(12):e70643. doi: 10.1111/jcmm.70643.
Neuronal PAS domain protein 2 (NPAS2) is critical in tissue fibrosis. Hypertrophic scars (HTS), a form of skin fibrosis, are characterised by excessive myofibroblast proliferation and abnormal extracellular matrix (ECM) deposition. However, whether NPAS2 contributes to skin fibrosis and the development of HTS remains unclear. In this study, the expression of NPAS2 between normal skin and hypertrophic scars (HTS) was assessed using RT-qPCR and immunohistochemistry (IHC). Human dermal fibroblasts (HDFs) and HTS-derived fibroblasts (HTS-Fs) were isolated from normal skin and HTS, respectively. NPAS2 was knocked down in HTS-Fs and overexpressed in HDFs via gene transfection. Cell proliferation and migration of transfected HTS-Fs and HDFs were analysed using flow cytometry, CCK-8 and transwell assays. The expressions of NPAS2, CLOCK, BMAL1, COL I, COL III, α-SMA and CDC25A were evaluated by western blotting and RT-qPCR. Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) identified the regulatory effect of NPAS2 on CDC25A. In vivo, an 8 × 8 mm full-thickness skin defect was created on the tail of SD rats, with viral particles (1 × 107) of r-plenR-sh-NPAS2 or r-plenR-NPAS2-NC injected subcutaneously at the wound edges weekly. Tissue samples, histopathological analyses and photographs were taken until the wound healed completely. The results indicated that NPAS2 was significantly upregulated in HTS. The proliferation, migration, and expression of COL I, COL III, and α-SMA were higher in HDFs overexpressing NPAS2 than those of HDFs themselves. In contrast, the behaviours mentioned above of HTS-Fs knocking down NPAS2 were lower than that of HTS-Fs. Mechanistically, the migration and proliferation promoting effect of NPAS2 was mediated by the binding of NPAS2 to the E-like-box of CDC25A. In vivo, compared with the r-plenR-NPAS2-NC group, the re-epithelialised regions of r-plenR-sh-NPAS2 were pink, flat and as large as the initial wound. In addition, their dermal structures were similar to skin and possessed loose and regular collagen arrangement which was parallel to the epidermis. Take together, these findings suggested that compared with HDFs, NPAS2 was upregulated in HTS-Fs. NPAS2 promoted the activation of HDFs, which is characterised by stronger proliferation and migration and the higher level of α-SMA, COL I and COL III. In which, the proliferation and migration effects of NPAS2 were mediated by CDC25A. Furthermore, NPAS2 knocked down in rat tail wounds inhibited the HTS formation. Therefore, NPAS2 may serve as a potential therapeutic target for HTS in the future.
神经元PAS结构域蛋白2（NPAS2）在组织纤维化中起关键作用。肥厚性瘢痕（HTS）是皮肤纤维化的一种形式，其特征在于肌成纤维细胞过度增殖和细胞外基质（ECM）异常沉积。然而，NPAS2是否促成皮肤纤维化和HTS的形成仍不清楚。在本研究中，使用RT-qPCR和免疫组织化学（IHC）评估正常皮肤和肥厚性瘢痕（HTS）中NPAS2的表达。人皮肤成纤维细胞（HDFs）和HTS来源的成纤维细胞（HTS-Fs）分别从正常皮肤和HTS中分离出来。通过基因转染在HTS-Fs中敲低NPAS2，并在HDFs中过表达NPAS2。使用流式细胞术、CCK-8和Transwell试验分析转染的HTS-Fs和HDFs的细胞增殖和迁移。通过蛋白质印迹和RT-qPCR评估NPAS2、CLOCK、BMAL1、COL I、COL III、α-SMA和CDC25A的表达。双荧光素酶报告基因试验和染色质免疫沉淀（ChIP）确定了NPAS2对CDC25A的调节作用。在体内，在SD大鼠尾部制造一个8×8mm的全层皮肤缺损，每周在伤口边缘皮下注射r-plenR-sh-NPAS2或r-plenR-NPAS2-NC的病毒颗粒（1×107）。采集组织样本、进行组织病理学分析并拍照，直到伤口完全愈合。结果表明，NPAS2在HTS中显著上调。过表达NPAS2的HDFs中COL I、COL III和α-SMA的增殖、迁移及表达高于HDFs本身。相反，敲低NPAS2的HTS-Fs的上述行为低于HTS-Fs。机制上，NPAS2的迁移和增殖促进作用是由NPAS2与CDC25A的E样框结合介导的。在体内，与r-plenR-NPAS2-NC组相比，r-plenR-sh-NPAS2组的再上皮化区域呈粉红色、平坦，与初始伤口大小相同。此外，它们的真皮结构类似于皮肤，胶原排列疏松且规则，与表皮平行。综上所述，这些发现表明，与HDFs相比，NPAS2在HTS-Fs中上调。NPAS促HDFs的激活，其特征是更强的增殖和迁移以及更高水平的α-SMA、COL I和COL III。其中，NPAS2的增殖和迁移作用由CDC25A介导。此外，在大鼠尾部伤口中敲低NPAS2可抑制HTS的形成。因此，NPAS2未来可能作为HTS的潜在治疗靶点。
